CN101633742B - Aminoplast substrate and its preparation method and application - Google Patents
Aminoplast substrate and its preparation method and application Download PDFInfo
- Publication number
- CN101633742B CN101633742B CN2009100921462A CN200910092146A CN101633742B CN 101633742 B CN101633742 B CN 101633742B CN 2009100921462 A CN2009100921462 A CN 2009100921462A CN 200910092146 A CN200910092146 A CN 200910092146A CN 101633742 B CN101633742 B CN 101633742B
- Authority
- CN
- China
- Prior art keywords
- amino
- substrate
- solution
- chitosan
- plastic
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Landscapes
- Other Resins Obtained By Reactions Not Involving Carbon-To-Carbon Unsaturated Bonds (AREA)
- Treatments Of Macromolecular Shaped Articles (AREA)
Abstract
The invention discloses an aminoplast substrate and a preparation method and application thereof. The method for preparing the biochip substrate comprises the following steps: and (3) connecting amino groups on the surface of the plastic sheet base to obtain the substrate with the amino groups connected on the surface. Experiments prove that the invention realizes the preparation of the biochip with excellent performance on the plastic material. The plastic has strong plasticity, is convenient for molding and processing, and can be applied to the field of biological chips such as micro-fluidic chips and the like because various microstructures and functional units are designed.
Description
Technical field
The present invention relates to amino plastic substrate and preparation method thereof and application.
Background technology
The current chip technology has become and has carried out a kind of vital technology extensive, high-throughput research in the biology.Along with the development of biochip technology, material technology, the surface chemistry technology relevant with its preparation also need constantly be improved and improve, and this embodies a concentrated reflection of in the preparation of bio-chip substrate of differing materials.At present mainly carry out the preparation of bio-chip substrate with inflexible inorganic materials-glass; But by the substrate that glass is processed be mainly used in to respond all be the preparation of the passive type biochip that carries out through the mode of STOCHASTIC DIFFUSION, and seldom adopting aspect the active chip that can reach accurate operation molecule and cell and control reaction purpose.Compare plastic material, glass material is difficult for machine-shaping, and this makes it particularly unable to do what one wishes in the field such as micro-fluidic biochip such as grade that is the basis with various microstructures.
Summary of the invention
An object of the present invention is to provide substrate of a kind of biochip and preparation method thereof.
The method for preparing bio-chip substrate provided by the present invention comprises the steps: to connect amino on the plastic base surface, obtains the surface connection and goes up amino substrate.
The method that connects described in the said process can be the method that comprises the steps: said plastic base is carried out the surface oxidation modification; Again with aminosilane reagent generation Silanization reaction; Make to form carbon-oxygen-silicon covalent linkage between aminosilane reagent and plastic base and be connected, obtain the surface and connect and go up amino substrate.For the ease of statement, this method note is made method I.
Among the aforesaid method I; Said said plastic base is carried out the surface oxidation modification; Method with aminosilane reagent generation Silanization reaction can comprise the steps: to place the solution of aminosilane reagent with after the modification of said plastic base surface oxidation again, carries out Silanization reaction.
Among above-mentioned arbitrary said method I, the concentration of aminosilane reagent can be 0.01%-10% (volumn concentration) in the solution of said aminosilane reagent.
Among above-mentioned arbitrary said method I, said aminosilane reagent can be (3-aminopropyl) triethoxysilane (be called for short APTS), (3-aminopropyl) diethoxymethylsilane (being called for short APDMS) or (3-aminopropyl) ethoxydimethylsilane (being called for short APEMS).
Among above-mentioned arbitrary said method I, the solvent in the solution of said aminosilane reagent can be water or ethanol.
Among above-mentioned arbitrary said method I, said said plastic base is carried out the surface oxidation modification, the method with aminosilane reagent generation Silanization reaction specifically can be following 1 again), 2) or 3):
1) said plastic base is polymethylmethacrylate sheet base, POLYCARBONATE SHEET base or polystyrene sheet base, and the solvent in the said aminosilane reagent solution is a water, and the temperature of said Silanization reaction is 15 ℃-70 ℃;
2) said plastic base is POLYCARBONATE SHEET base or polystyrene sheet base, and the solvent in the said aminosilane reagent solution is an ethanol, and the temperature of said Silanization reaction is 15 ℃-70 ℃;
3) said plastic base is a polymethylmethacrylate sheet base, and the solvent in the said aminosilane reagent solution is an ethanol, and the temperature of said Silanization reaction is 15 ℃-30 ℃.
Connecting the substrate that obtains through above-mentioned arbitrary said method I is two-dimentional amino plastic substrate.
The method that connects described in the said process can be the method that comprises the steps: after said plastic base is carried out the surface oxidation modification, place the solution that is rich in amino polymkeric substance, absorption obtains the surface and is connected with amino substrate.This method is actual to be realized through electrostatic adhesion.For the ease of statement, this method note is made method II.
Among the aforesaid method II, the temperature of said absorption can be 10 ℃-50 ℃.
Among above-mentioned arbitrary said method II, the time of said absorption can be 0.5-30 hour.
Among above-mentioned arbitrary said method II, actual is to realize through the electrostatic adsorption of being rich in amino polymer molecule and plastic base surface.Before placing the solution that is rich in amino polymkeric substance; Earlier plastic base is cleaned up; Then plastic base is carried out under ozonize or the logical oxygen condition Plasma and handle to reach required hydrophilic requirement and surface oxidation modification, the treatment time changes with hydrophilic requirement.
The method that connects described in the said process also can be the method that comprises the steps: with said plastic base epoxy groupization, obtain the plastic base of epoxy groupization; The plastic base of said epoxy groupization is placed the solution that is rich in amino polymkeric substance, and graft reaction obtains the surface and is connected with amino substrate.For the ease of statement, this method note is made method III.
Among the aforesaid method III; Said method with said plastic base epoxy groupization can comprise the steps: said plastic base is carried out the surface oxidation modification earlier; Place the solution of epoxy radicals silicone hydride reagent then, the epoxy glycosylation reaction obtains the plastic base of said epoxy groupization.
Among above-mentioned arbitrary said method III, the solvent in the solution of said epoxy radicals silicone hydride reagent can be water or ethanol.
Among above-mentioned arbitrary said method III, the concentration of epoxy radicals silicone hydride reagent can be 0.01%-10% (volumn concentration) in the solution of said epoxy radicals silicone hydride reagent.
Among above-mentioned arbitrary said method III, the time of said epoxy glycosylation reaction can be 0.5-30 hour.
Among above-mentioned arbitrary said method III, said method with the plastic base epoxy groupization specifically can be as following 1), 2) or 3):
1) said plastic base is polymethylmethacrylate sheet base, POLYCARBONATE SHEET base or polystyrene sheet base, and the solvent in the said epoxy radicals silicone hydride reagent solution is a water, and the temperature of said epoxy glycosylation reaction is 15 ℃-70 ℃;
2) said plastic base is POLYCARBONATE SHEET base or polystyrene sheet base, and the solvent in the said epoxy radicals silicone hydride reagent solution is an ethanol, and the temperature of said epoxy glycosylation reaction is 15 ℃-70 ℃;
3) said plastic base is a polymethylmethacrylate sheet base, and the solvent in the said epoxy radicals silicone hydride reagent solution is an ethanol, and the temperature of said epoxy glycosylation reaction is 15 ℃-30 ℃.
Among above-mentioned arbitrary said method III, said epoxy radicals silicone hydride reagent can be (3-Glycidyloxypropyl) trimethoxysilane (be called for short GPTMS), (3-Glycidoxypropyl) dimethylethoxysilane (being called for short GPDMES) or (3-Glycidoxypropyl) dimethoxymethylsilane (being called for short GPDMMS).
Among above-mentioned arbitrary said method III, the temperature of said graft reaction is 10-50 ℃.
Among above-mentioned arbitrary said method III, the time of said graft reaction is 0.5-30 hour.
Among above-mentioned arbitrary said method II and the method III, the said solution that is rich in amino polymkeric substance is water.
Among above-mentioned arbitrary said method II and the method III, the concentration that is rich in amino polymkeric substance in the said solution that is rich in amino polymkeric substance is 0.001%-0.3% (quality percentage composition).
Among above-mentioned arbitrary said method II and the method III, the said polymkeric substance that is rich in amino includes but not limited to chitosan, poly-lysine, polyethylene imine based or PAH.
In above-mentioned arbitrary said method; When the said polymkeric substance that is rich in amino is chitosan; The solution of said chitosan is to make according to the method that comprises the steps: use earlier the acetate dissolution chitosan; Obtain chitosan solution I, use 0.1 * PBS damping fluid to dilute said chitosan solution I again, obtain the solution of said chitosan; The pH value of the solution of said chitosan is 5.0-6.0.
In above-mentioned arbitrary said method, said plastic base includes but not limited to POLYCARBONATE SHEET base, polystyrene sheet base or polymethylmethacrylate sheet base.
Among above-mentioned arbitrary said method I, method II and the method III, saidly plastic base is carried out oxidizing methods for surface modification of color can comprise the steps: said plastic base is handled said plastic base Plasma with ozonize or under logical oxygen condition.
The bio-chip substrate that is prepared by above-mentioned arbitrary said method also belongs to protection scope of the present invention.
The biochip that is made by above-mentioned arbitrary said substrate also belongs to protection scope of the present invention.
Substrate of the present invention is a kind of amido modified plastic substrate; Handle through the surface oxidation modification earlier specifically; Directly aminosilaneization makes two-dimentional amino plastic substrate then; Thereby also can directly be coated with to apply and be rich in amino polymer polymer formation three-dimensional amino-group plastic substrate through electrostatic adhesion; Can also select diverse ways on plastic base, to carry out earlier surface epoxy group modified (promptly preparing the epoxy group(ing) plastic substrate),, finally obtain the surface and be the polymer three-dimensional amino plastic substrate then through being rich in amino polymkeric substance polymer in the grafting of epoxy group(ing) reactive group.The two kinds of methods in back are owing to adopt the polymkeric substance polymer that is rich in amino; Its long hydrophilic connecting arm has enough suppleness; The sample molecule that is fixed in substrate surface is fully contacted with target molecule; Very fast combination and the balance that realizes between the molecule, thus the stability of substrate improved, and fixedly sensitivity is improved simultaneously.The experiment proof; That the surface of amino plastic substrate of the present invention explanation plastic material can be carried out is well amido modified (even can by amino extend aldehyde group modified etc.) and epoxy group modified; And then process amino plastic substrate, realized the good biochip of processability on plastic material.Because plastic plasticity is strong, be easy to forming process, can prepare various microstructures, various functional unit, so substrate of the present invention will have broad application prospects in biochip field such as micro-fluidic.
Description of drawings
Fig. 1 nucleic acid chip dot matrix schema.
The chip hybridization design sketch of the common amino-group substrate point system of Fig. 2 Oligo.
Fig. 3 is the chip hybridization design sketch that the two-dimentional amino plastic substrate (substrates of embodiment 3 preparations) through the aminosilane preparation is gone up some system Oligo.
Fig. 4 is a chip hybridization design sketch of going up some system Oligo through the three-dimensional amino-group plastic substrate of direct coating preparation (substrates of embodiment 12 preparations).
Fig. 5 is a chip hybridization design sketch of going up some system Oligo through the three-dimensional amino-group plastic substrate of covalent attachment preparation (substrates of embodiment 15 preparations).
Fig. 6 is a chip hybridization design sketch of going up some system Oligo through the three-dimensional amino-group plastic substrate of covalent attachment preparation (substrates of embodiment 16 preparations).
Embodiment
Employed experimental technique is ordinary method like no specified otherwise among the following embodiment.
Used material, reagent etc. like no specified otherwise, all can obtain from commercial sources among the following embodiment.
Chitosan (chitosan), poly-lysine (polylysine), polyethylene imine based (polyethyleneimine), PAH (polyallylamine) are all available from Sigma.
(3-Glycidyloxypropyl) trimethoxysilane (be called for short GPTMS); (3-Glycidoxypropyl) dimethylethoxysilane (be called for short GPDMES), (3-Glycidoxypropyl) dimethoxymethylsilane (being called for short GPDMMS),
(3-aminopropyl) triethoxysilane (be called for short APTS), (3-aminopropyl) diethoxymethylsilane (being called for short APDMS) and (3-aminopropyl) ethoxydimethylsi lane (being called for short APEMS) available from Sigma.
POLYCARBONATE SHEET base (being called for short PC sheet base), its material is polycarbonate (Polycarbonate);
Polystyrene sheet base (being called for short PS sheet base), its material is PS (Polystyrene), PS is meant by styrene monomer through radical polycondensation synthetic polymkeric substance;
Polymethylmethacrylate sheet base (being called for short PMMA sheet base), its material is polymethylmethacrylate (Polymethylmethacrylate).
Above PC sheet base, PS sheet base and PMMA sheet base be all available from Beijing east pearl synthetic glass business department, and size is (75.5mm ± 0.2mm) * (25.2mm ± 0.2mm) * (1.0mm).
Chitosan solution among the following embodiment all makes according to following method: earlier with 1% (volume percent) acetate dissolution chitosan; Obtain strong solution; Use 0.1 * PBS damping fluid to dilute said strong solution then, obtain chitosan solution, and the pH value of adjusting chitosan solution is to 5.0-6.0.
1, the pre-treatment of sheet base:, carry out O with plastic base PS washed with de-ionized water, drying
3Handle Plasma processing 2min under 3h or the logical oxygen condition.
2, aminosilaneization: with the pretreated plastic base of step 1, place the aqueous solution of APTS, the concentration of APTS in solution is 0.01% (volumn concentration), shakes under the condition reaction 30 hours at 70 ℃, 120rpm; Promptly get two-dimentional amino plastic substrate with washed with de-ionized water, drying.
Plastic base is through surface cleaning and surface-treated, thereby plastic base is surperficial to distribute for amino through making with the APTS reaction, makes amino plastic substrate (two-dimentional amino plastic substrate).
Change plastic base PS into PC or PMMA, carry out above-mentioned experiment, also obtained two-dimentional amino plastic substrate.
Embodiment 2, in the aqueous solution, prepare two-dimentional amino plastic substrate with the aminosilane method
1, the pre-treatment of sheet base:
With testing 1 among the embodiment 1.
2, aminosilaneization: with the pretreated plastic base of step 1, place the aqueous solution of APTS, the concentration of APTS in solution is 10% (volumn concentration), shakes under the condition reaction 0.5 hour at 15 ℃, 120rpm; Promptly get two-dimentional amino plastic substrate with washed with de-ionized water, drying.
Change plastic base PS into PC or PMMA, carry out above-mentioned experiment, also obtained two-dimentional amino plastic substrate.
Embodiment 3, in the aqueous solution, prepare two-dimentional amino plastic substrate with the aminosilane method
1, the pre-treatment of sheet base:
With testing 1 among the embodiment 1.
2, aminosilaneization: with the pretreated plastic base of step 1, place the aqueous solution of APTS, the concentration of APTS in solution is 0.5% (volumn concentration), shakes under the condition reaction 5 hours at 35 ℃, 120rpm; Promptly get two-dimentional amino plastic substrate with washed with de-ionized water, drying.
Change plastic base PS into PC or PMMA, carry out above-mentioned experiment, also obtained two-dimentional amino plastic substrate.
Embodiment 4, in ethanolic soln, prepare two-dimentional amino plastic substrate with the aminosilane method
1. the pre-treatment of sheet base:, carry out O with plastic base PC washed with de-ionized water, drying
3Handle Plasma processing 3min under 5h or the logical oxygen condition.
2, aminosilaneization: with the pretreated plastic base of step 1, place the ethanolic soln of APDMS, the concentration of APDMS in solution is 10% (volumn concentration), shakes under the condition reaction 0.3 hour at 15 ℃, 120rpm; Promptly get amino plastic substrate (two-dimentional amino plastic substrate) with ethanol cleaning, drying.
PC changes PS into plastic substrate, carries out above-mentioned experiment, has also obtained two-dimentional amino plastic substrate.
Embodiment 5, in ethanolic soln, prepare two-dimentional amino plastic substrate with the aminosilane method
1. the pre-treatment of sheet base:
With testing 1 among the embodiment 4.
2, aminosilaneization: with the pretreated plastic base of step 1, place the ethanolic soln of APDMS, the concentration of APDMS in solution is 0.01% (volumn concentration), shakes under the condition reaction 30 hours at 70 ℃, 120rpm; Promptly get amino plastic substrate (two-dimentional amino plastic substrate) with ethanol cleaning, drying.
PC changes PS into plastic substrate, carries out above-mentioned experiment, has also obtained two-dimentional amino plastic substrate.
Embodiment 6, in ethanolic soln, prepare two-dimentional amino plastic substrate with the aminosilane method
1. the pre-treatment of sheet base:
With testing 1 among the embodiment 4.
2, aminosilaneization: with the pretreated plastic base of step 1, place the ethanolic soln of APDMS, the concentration of APDMS in solution is 0.5% (volumn concentration), shakes under the condition reaction 5 hours at 45 ℃, 120rpm; Promptly get amino plastic substrate (two-dimentional amino plastic substrate) with ethanol cleaning, drying.
PC changes PS into plastic substrate, carries out above-mentioned experiment, has also obtained two-dimentional amino plastic substrate.
Embodiment 7, in ethanolic soln, prepare two-dimentional amino plastic substrate with the aminosilane method
1, the pre-treatment of sheet base:, carry out O with plastic base PMMA washed with de-ionized water, drying
3Handle Plasma processing 1min under 2h or the logical oxygen condition.
2, aminosilaneization: with the pretreated plastic base of step 1, place the ethanolic soln of APEMS, the concentration of APEMS in solution is 0.5% (volumn concentration), shakes under the condition reaction 15 hours at 20 ℃, 120rpm; Promptly get amino plastic substrate (two-dimentional amino plastic substrate) with ethanol cleaning, drying.
Embodiment 8, in ethanolic soln, prepare two-dimentional amino plastic substrate with the aminosilane method
1, the pre-treatment of sheet base:
With testing 1 among the embodiment 7.PMMA experimentizes with plastic base.
2, aminosilaneization: with the pretreated plastic base of step 1, place the ethanolic soln of APEMS, the concentration of APEMS in solution is 10% (volumn concentration), shakes under the condition reaction 0.5 hour at 30 ℃, 120rpm; Promptly get amino plastic substrate (two-dimentional amino plastic substrate) with ethanol cleaning, drying.
Embodiment 9, in ethanolic soln, prepare two-dimentional amino plastic substrate with the aminosilane method
1, the pre-treatment of sheet base:
With testing 1 among the embodiment 7.PMMA experimentizes with plastic base.
2, aminosilaneization: with the pretreated plastic base of step 1, place the ethanolic soln of APEMS, the concentration of APEMS in solution is 0.01% (volumn concentration), shakes under the condition reaction 30 hours at 15 ℃, 120rpm; Promptly get amino plastic substrate (two-dimentional amino plastic substrate) with ethanol cleaning, drying.
Experiment shows, PMMA sheet base ethanol mutually in, the swelling phenomenon can occur under the condition of temperature higher (as 45 ℃, 50 ℃, 70 ℃), and then cause the plastic base distortion, can not get qualified two-dimentional amino plastic substrate.
Embodiment 10, on plastic base, directly apply and be rich in amino polymkeric substance polymer, preparation surface three dimension amino plastic substrate
1, the cleaning of plastic base and surface oxidation modification
After plastic base PMMA cleaned up with deionized water, handle 2min with ozonize 3h or with Plasma under the logical oxygen condition;
2, apply the polymkeric substance polymer that is rich in amino
Chitosan (chitosan) solution: for final concentration is the 0.1XPBS damping fluid of the chitosan of 0.001% (quality percentage composition), said final concentration is the concentration of chitosan in chitosan solution; It is 6.0 that the pH value of chitosan solution transfers to.
The surface-treated plastic base that step 1 is obtained immerses in chitosan (chitosan) solution, and reaction is 0.5 hour under 50 ℃, the condition of shaking with the speed of 120rpm; Stopped reaction, with plastic base with deionized water wash, drying.Obtain the surface three dimension amino plastic substrate.
Plastic base PMMA is replaced with PC or PS; Again chitosan is replaced with the polymkeric substance polylysine, polyethyleneimine or the polyallylamine that are rich in amino respectively and carry out above-mentioned experiment; The solution of different is these three kinds of polymkeric substance is the aqueous solution, and the result has all obtained the three-dimensional amino-group plastic substrate.
After plastic sheet passes through surface cleaning and surface oxidation modification; Thereby part on the amino polymkeric substance polymer is amino electrostatic adsorption takes place to be made and is rich in the amino polymer polymer on the plastic base surface adsorption with being rich in; Each is rich in amino remaining on the amino polymer molecule makes the plastic base surface form three-dimensional amino, i.e. the amino amino plastic substrate (three-dimensional amino-group plastic substrate) that distributes of surface three dimension.
Embodiment 11, on plastic base, directly apply and be rich in amino polymkeric substance polymer, preparation surface three dimension amino plastic substrate
1, the cleaning of plastic base and surface oxidation modification
With testing 1 among the embodiment 10.
2, apply the polymkeric substance polymer that is rich in amino
Chitosan (chitosan) solution: for final concentration is the 0.1XPBS damping fluid of the chitosan of 0.3% (quality percentage composition), said final concentration is the concentration of chitosan in chitosan solution; It is 5.0 that the pH value of chitosan solution transfers to.
The surface-treated plastic base that step 1 is obtained immerses in chitosan (chitosan) solution, and reaction is 30 hours under 10 ℃, the condition of shaking with the speed of 120rpm; Stopped reaction, with plastic base with deionized water wash, drying.Obtain the surface three dimension amino plastic substrate.
Plastic base PMMA is replaced with PC or PS; Again chitosan is replaced with the polymkeric substance polylysine, polyethyleneimine or the polyallylamine that are rich in amino respectively and carry out above-mentioned experiment; The solution of different is these three kinds of polymkeric substance is the aqueous solution, and the result has all obtained the three-dimensional amino-group plastic substrate.
Embodiment 12, on plastic base, directly apply and be rich in amino polymkeric substance polymer, preparation surface three dimension amino plastic substrate
1, the cleaning of plastic base and surface oxidation modification
With testing 1 among the embodiment 10.
2, apply the polymkeric substance polymer that is rich in amino
Chitosan (chitosan) solution: for final concentration is the 0.1XPBS damping fluid of the chitosan of 0.06% (quality percentage composition), said final concentration is the concentration of chitosan in chitosan solution; It is 5.5 that the pH value of chitosan solution transfers to.
The surface-treated plastic base that step 1 is obtained immerses in chitosan (chitosan) solution, and reaction is 15 hours under 25 ℃, the condition of shaking with the speed of 120rpm; Stopped reaction, with plastic base with deionized water wash, drying.Obtain the surface three dimension amino plastic substrate.
Plastic base PMMA is replaced with PC or PS; Again chitosan is replaced with the polymkeric substance polylysine, polyethyleneimine or the polyallylamine that are rich in amino respectively and carry out above-mentioned experiment; The solution of different is these three kinds of polymkeric substance is the aqueous solution, and the result has all obtained the three-dimensional amino-group plastic substrate.
Embodiment 13, in the aqueous solution, prepare the plastic base of epoxy groupization, prepare the three-dimensional amino-group plastic substrate with this sheet base again with chemical process
1, the pre-treatment of sheet base
Plastic base PC is carried out ozonize or Plasma processing, and concrete grammar is after plastic base is cleaned, dries, to carry out Plasma processing 3min under surperficial ozonize 5h or the logical oxygen condition.
2, epoxy groupization also promptly prepares the epoxy plastics substrate
The pretreated plastic base of step 1 is placed the aqueous solution of GPTMS, and the concentration of GPTMS in solution is 0.01% (volumn concentration), shakes under the condition reaction 0.5 hour at 70 ℃, 300rpm; With washed with de-ionized water, drying;
3, amino polymkeric substance polymer is rich in grafting
Polyallylamine solution: for final concentration is the aqueous solution of the polyallylamine of 0.001% (quality percentage composition), said final concentration is the concentration of polyallylamine in polyallylamine solution.
The plastic base of the epoxy groupization that step 2 is obtained also is that the epoxy plastics substrate immerses in the polyallylamine solution, and reaction is 0.5 hour under 50 ℃, the condition of shaking with the speed of 120rpm.
Stopped reaction, with plastic base with deionized water wash, drying.Obtain three-dimensional amino-group plastic substrate (three-dimensional amino-group plastic substrate).
Plastic base PC is replaced with PS or PMMA, and the polymkeric substance polyallylamine that will be rich in amino again replaces with chitosan, polylysine or polyethyleneimine and carries out above-mentioned experiment, has also all obtained the three-dimensional amino-group plastic substrate.Different is, chitosan (chitosan) solution is that final concentration is the 0.1X PBS damping fluid of the chitosan of 0.001% (quality percentage composition), and said final concentration is the concentration of chitosan in chitosan solution; It is 6.0 that the pH value of chitosan solution transfers to.
Embodiment 14, in the aqueous solution, prepare the plastic base of epoxy groupization, prepare the three-dimensional amino-group plastic substrate with this sheet base again with chemical process
1, the pre-treatment of sheet base
With testing 1 among the embodiment 13.
2, epoxy groupization also promptly prepares the epoxy plastics substrate
The pretreated plastic base of step 1 is placed the aqueous solution of GPTMS, and the concentration of GPTMS in solution is 10% (volumn concentration), shakes under the condition reaction 30 hours at 15 ℃, 300rpm; With washed with de-ionized water, drying;
3, amino polymkeric substance polymer is rich in grafting
Polyallylamine solution: for final concentration is the aqueous solution of the polyallylamine of 0.3% (quality percentage composition), said final concentration is the concentration of polyallylamine in polyallylamine solution.
The plastic base of the epoxy groupization that step 2 is obtained also is that the epoxy plastics substrate immerses in the polyallylamine solution, and reaction is 30 hours under 10 ℃, the condition of shaking with the speed of 120rpm.
Stopped reaction, with plastic base with deionized water wash, drying.Obtain the amino amino plastic substrate (three-dimensional amino-group plastic substrate) of surface three dimension.
Plastic base PC is replaced with PS or PMMA, and the polymkeric substance polyallylamine that will be rich in amino again replaces with chitosan, polylysine or polyethyleneimine and carries out above-mentioned experiment, has also all obtained the three-dimensional amino-group plastic substrate.Different is, chitosan (chitosan) solution is that final concentration is the 0.1X PBS damping fluid of the chitosan of 0.3% (quality percentage composition), and said final concentration is the concentration of chitosan in chitosan solution; It is 5.0 that the pH value of chitosan solution transfers to.
Embodiment 15, in the aqueous solution, prepare the plastic base of epoxy groupization, prepare the three-dimensional amino-group plastic substrate with this sheet base again with chemical process
1, the pre-treatment of sheet base
With testing 1 among the embodiment 13.
2, epoxy groupization also promptly prepares the epoxy plastics substrate
The pretreated plastic base of step 1 is placed the aqueous solution of GPTMS, and the concentration of GPTMS in solution is 0.5% (volumn concentration), shakes under the condition reaction 15 hours at 35 ℃, 300rpm; With washed with de-ionized water, drying;
3, amino polymkeric substance polymer is rich in grafting
Polyallylamine solution: for final concentration is the aqueous solution of the polyallylamine of 0.06% (quality percentage composition), said final concentration is the concentration of polyallylamine in polyallylamine solution.
The plastic base of the epoxy groupization that step 2 is obtained also is that the epoxy plastics substrate immerses in the polyallylamine solution, and reaction is 15 hours under 25 ℃, the condition of shaking with the speed of 120rpm.
Stopped reaction, with plastic base with deionized water wash, drying.Obtain the surface and be the amino plastic substrate of three-dimensional amino-group (three-dimensional amino-group plastic substrate).
Plastic base PC is replaced with PS or PMMA, and the polymkeric substance polyallylamine that will be rich in amino again replaces with chitosan, polylysine or polyethyleneimine and carries out above-mentioned experiment, has also all obtained the three-dimensional amino-group plastic substrate.Different is, chitosan (chitosan) solution is that final concentration is the 0.1X PBS damping fluid of the chitosan of 0.06% (quality percentage composition), and said final concentration is the concentration of chitosan in chitosan solution; It is 5.5 that the pH value of chitosan solution transfers to.
Embodiment 16, in ethanolic soln, prepare the plastic base of epoxy groupization, prepare the three-dimensional amino-group plastic substrate with this sheet base again with chemical process
1, the pre-treatment of plastic base
With testing 1 among the embodiment 13.
2, plastic base PC epoxy groupization also promptly prepares the epoxy plastics substrate: the ethanolic soln that the pretreated plastic base of step 1 is placed GPDMES; The concentration of GPDMES in solution is 10% (volumn concentration), shakes under the condition reaction 0.5 hour at 15 ℃, 150rpm; Clean, dry with ethanol;
3, amino polymkeric substance polymer is rich in grafting
Chitosan (chitosan) solution: for final concentration is the 0.1XPBS damping fluid of the chitosan of 0.08% (quality percentage composition), said final concentration is the concentration of chitosan in chitosan solution; It is 6.0 that the pH value of chitosan solution transfers to.
The plastic base of the epoxy groupization that step 2 is obtained immerses in chitosan (chitosan) solution, and reaction is 3 hours under 25 ℃, the condition of shaking with the speed of 120rpm; Stopped reaction, with plastic base with deionized water wash, drying.Obtain the surface three dimension amino plastic substrate.
PC replaces with PS with plastic base; The polymkeric substance chitosan that will be rich in amino again replaces with polylysine, polyallylamine or polyethyleneimine and carries out above-mentioned experiment; The solution of different is these three kinds of polymkeric substance is the aqueous solution, and the result has also all obtained the three-dimensional amino-group plastic substrate.
Embodiment 17, in ethanolic soln, prepare the plastic base of epoxy groupization, prepare the three-dimensional amino-group plastic substrate with this sheet base again with chemical process
1, the pre-treatment of plastic base
With testing 1 among the embodiment 13.
2, plastic base PC epoxy groupization also promptly prepares the epoxy plastics substrate: the ethanolic soln that the pretreated plastic base of step 1 is placed GPDMES; The concentration of GPDMES in solution is 0.01% (volumn concentration), shakes under the condition reaction 0.5 hour at 70 ℃, 150rpm; Clean, dry with ethanol;
3, amino polymkeric substance polymer is rich in grafting
With the experiment among the embodiment 16 3.
Clean, dry, promptly get the three-dimensional amino-group plastic substrate.
PC replaces with PS with plastic base; The polymkeric substance chitosan that will be rich in amino again replaces with polylysine, polyallylamine or polyethyleneimine and carries out above-mentioned experiment; The solution of different is these three kinds of polymkeric substance is the aqueous solution, and the result has also all obtained the three-dimensional amino-group plastic substrate.
Embodiment 18, in ethanolic soln, prepare the plastic base of epoxy groupization, prepare the three-dimensional amino-group plastic substrate with this sheet base again with chemical process
1, the pre-treatment of plastic base
With testing 1 among the embodiment 13.
2, plastic base PC epoxy groupization also promptly prepares the epoxy plastics substrate: the ethanolic soln that the pretreated plastic base of step 1 is placed GPDMES; The concentration of GPDMES in solution is 0.5% (volumn concentration), shakes under the condition reaction 3 hours at 50 ℃, 150rpm; Clean, dry with ethanol;
3, amino polymkeric substance polymer is rich in grafting
With the experiment among the embodiment 16 3.
Clean, dry, promptly get the three-dimensional amino-group plastic substrate.
PC replaces with PS with plastic base; The polymkeric substance chitosan that will be rich in amino again replaces with polylysine, polyallylamine or polyethyleneimine and carries out above-mentioned experiment; The solution of different is these three kinds of polymkeric substance is the aqueous solution, and the result has also all obtained the three-dimensional amino-group plastic substrate.
Embodiment 19, in ethanolic soln, prepare the plastic base of epoxy groupization, prepare the three-dimensional amino-group plastic substrate with this sheet base again with chemical process
1, the pre-treatment of plastic base
PMMA experimentizes with plastic base.Treatment process is with testing 1 among the embodiment 13.
2, plastics PMMA sheet base epoxy groupization also promptly prepares the epoxy plastics substrate: the ethanolic soln that the pretreated plastic base of step 1 is placed GPDMMS; The concentration of GPDMMS in solution is 10% (volumn concentration), shakes under the condition reaction 30 hours at 15 ℃, 150rpm; Clean, dry with ethanol;
3, amino polymkeric substance polymer is rich in grafting
Polylysine solution: for final concentration is the aqueous solution of the polylysine of 0.08% (quality percentage composition), said final concentration is the concentration of polylysine in polylysine solution.
The plastic base of the epoxy groupization that step 2 is obtained immerses in the polylysine solution, and reaction is 30 hours under 25 ℃, the condition of shaking with the speed of 120rpm; Stopped reaction, with plastic base with deionized water wash, drying.Obtain surface three dimension aminoplastics PMMA substrate.
The polymkeric substance polylysine that is rich in amino is replaced with chitosan, polyallylamine or polyethyleneimine carry out above-mentioned experiment, also all obtained three-dimensional amino-group plastics PMMA substrate.Different is, chitosan (chitosan) solution is that final concentration is the 0.1X PBS damping fluid of the chitosan of 0.08% (quality percentage composition), and said final concentration is the concentration of chitosan in chitosan solution; It is 6.0 that the pH value of chitosan solution transfers to.
Embodiment 20, in ethanolic soln, prepare the plastic base of epoxy groupization, prepare the three-dimensional amino-group plastic substrate with this sheet base again with chemical process
1, the pre-treatment of plastic base
PMMA experimentizes with plastic base.Treatment process is with testing 1 among the embodiment 13.
2, plastics PMMA sheet base epoxy groupization also promptly prepares the epoxy plastics substrate: the ethanolic soln that the pretreated plastic base of step 1 is placed GPDMMS; The concentration of GPDMMS in solution is 0.01% (volumn concentration), shakes under the condition reaction 0.5 hour at 30 ℃, 150rpm; Clean, dry with ethanol;
3, amino polymkeric substance polymer is rich in grafting
With the experiment among the embodiment 19 3.
Clean, dry, promptly get three-dimensional amino-group plastics PMMA substrate.
The polymkeric substance polylysine that is rich in amino is replaced with chitosan, polyallylamine or polyethyleneimine carry out above-mentioned experiment, also all obtained three-dimensional amino-group plastics PMMA substrate.Different is, chitosan (chitosan) solution is that final concentration is the 0.1X PBS damping fluid of the chitosan of 0.08% (quality percentage composition), and said final concentration is the concentration of chitosan in chitosan solution; It is 5.5 that the pH value of chitosan solution transfers to.
Experiment shows, PMMA sheet base ethanol mutually in, as the swelling phenomenon can occurring under 45 ℃, 50 ℃, 70 ℃ the condition, and then cause the plastic base distortion when temperature is higher, can not get qualified three-dimensional amino-group plastic substrate.
By the amino plastic substrate surface molecular similar that embodiment 1-9 makes, substrate each to put up be the amino plastic substrate of an amino, the whole sheet base is modified to two-dimentional amino plastic substrate; The surface molecular similar of the substrate that makes by embodiment 10-20; Each puts up substrate and is one and contains several, tens or the long chain polymer of polyamino more, so the whole sheet primary surface forms the three-dimensional amino-group plastic substrate that a kind of high-polymer three-dimensional amino-group distributes.
Embodiment 21, common amino-group substrate and amino plastic substrate are applied to the preparation of nucleic acid chip
The used common amino-group substrate of present embodiment makes for slide aminosilane method commonly used at present, and promptly rebake is crosslinked after carry out aminosilaneization earlier on the slide, promptly gets common amino glass substrate.
The used amino plastic substrate of present embodiment is obtained by method among the embodiment 1-20 respectively.
The preparation process of biochip is following:
1. (its sequence is (T) with the 35mer Oligo sample of 3 kinds of different concns
15-TCGGATTCGACAACACCCGT contains 50%DMSO) solution, every kind of sample takes out 10uL and shifts in adding 384 orifice plates; Preparation 50%DMSO solution also takes out 10uL and shifts in adding 384 orifice plates as blank (BC) contrast; (positive quality control of point sample, its sequence are (T) to QC
15-GTG CAA CTC ACT CGA CTG-3 '-HEX; Contain 50%DMSO) be two kinds of concentration (1uM; 2.5uM) an end have same a kind of oligonucleotide probe of HEX mark, be used to observe chip point sample and fixed efficient, each takes out 10uL and shifts and add in 384 orifice plates.
2. through automatic spot sample device ready point sample sample is put respectively on common amino glass substrate and the amino plastic substrate surface by embodiment 3,12,15,16 preparations.Concrete dot matrix way is as shown in Figure 1: every chip comprises 4 dot matrix; Each dot matrix comprises 36 points; The dot matrix of each dot matrix is designed to 6 row *, 6 row; Sample repetition form is 1*6, and dot spacing is 400um, and 6 positions of sample on the chip dot matrix are followed successively by from top to bottom: 1uM QC, 2.5uM QC, BC, 2.5uM Oligo sample, 5.0uM Oligo sample, 10uM Oligo sample.
3. spend the night 37 ℃ of wet boxes of the chip behind the point sample fixing;
4. (its sequence is the chip hybridization liquid of HEX-5 '-ACTGGACTTGACGGGTGTTGTCGAATCCGA), under 100% humidity, in 42 ℃ of hybridization 12-16 hour down on each chip dot matrix, to add the Arab-HEX contain fluorescence HEX and to modify; Hybridization solution: 0.1%SDS, 3 * SSC, 25% methane amide, 5 * Denhardt ' s.
Reaction back chip respectively in two kinds of washing lotions 42 ℃ respectively wash 2min, washing lotion I:0.3 * SSC/0.1%SDS, washing lotion II:0.06 * SSC.At last, slide centrifuge dripping.
6. chip scanning and data processing.Brilliant core is used in data analysis
TMChip after Luxscan-10K/B (Boao Biological Co., Ltd) the scanning hybridization, sweep parameter is all set Laser/PMT=90/700.
Common amino glass substrate with by the scan image of the amino plastic substrate of embodiment 3,12,15,16 preparation respectively like Fig. 2,3,4,5, shown in 6.The two-dimentional plastic substrate that embodiment 1,2,4-9 make has the 3 similar effects with embodiment; The three-dimensional plastic substrate that embodiment 10 and 11 makes has the 12 similar effects with embodiment, and the three-dimensional plastic substrate that embodiment 13,14,17-20 make has and embodiment 15 or 16 similar effects.
Fig. 3 is result's ((a) two-dimentional amino PS substrate, (b) two-dimentional amino PC substrate, (c) two-dimentional amino PMMA substrate) of substrate among the embodiment 3;
Fig. 4 is that ((a) directly applies the three-dimensional amino-group PMMA substrate of Chitosan for the result of substrate among the embodiment 12; (b) directly apply the three-dimensional amino-group PS substrate of Polylysine; (c) directly apply the three-dimensional amino-group PS substrate of Polyallylamine; (d) directly apply the three-dimensional amino-group PC substrate of Chitosan, (e) directly apply the three-dimensional amino-group PS substrate of Chitosan).
Fig. 5 is result's (the three-dimensional amino-group PMMA substrate of (a) covalent attachment Chitosan, (b) the three-dimensional amino-group PS substrate of covalent attachment Polylysine, (c) three-dimensional amino-group PS substrate of covalent attachment Polyallylamine) of substrate among the embodiment 15.
Fig. 6 is result's (three-dimensional amino-group PS substrate of (a) covalent attachment Chitosan of substrate among the embodiment 16; (b) the three-dimensional amino-group PS substrate of covalent attachment Polylysine; (c) the three-dimensional amino-group PS substrate of covalent attachment Polyallylamine, (d) the three-dimensional amino-group PC substrate of covalent attachment Chitosan).
The result shows; Compare with common amino glass substrate; The present invention does not have significant difference by the amino plastic substrate of plastic base preparation for the nucleic acid samples fixed signal intensity of 3 kinds of different concns and the effect of common amino glass substrate, shows that substrate of the present invention can be used to prepare chip.Consider the characteristics that are easy to forming process of plastic material, of the present invention is that the amino-group substrate of material can be used in micro-fluidic the grade in the biochip field with plastics.
Claims (9)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009100921462A CN101633742B (en) | 2009-09-01 | 2009-09-01 | Aminoplast substrate and its preparation method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009100921462A CN101633742B (en) | 2009-09-01 | 2009-09-01 | Aminoplast substrate and its preparation method and application |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201110153777 Division CN102276863B (en) | 2009-09-01 | 2009-09-01 | Amino plastic substrate and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101633742A CN101633742A (en) | 2010-01-27 |
| CN101633742B true CN101633742B (en) | 2012-05-23 |
Family
ID=41593089
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2009100921462A Active CN101633742B (en) | 2009-09-01 | 2009-09-01 | Aminoplast substrate and its preparation method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101633742B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106513068B (en) * | 2016-10-25 | 2018-10-30 | 清华大学 | The solution and its application that micro-fluidic chip bonding for polymerizable material is modified with surface |
| CN108085314B (en) * | 2016-11-21 | 2021-11-09 | 杭州梓晶生物有限公司 | Aminated filter paper/membrane for nucleic acid extraction and purification and preparation method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101241124A (en) * | 2008-02-18 | 2008-08-13 | 博奥生物有限公司 | A kind of biochip substrate and preparation method thereof |
-
2009
- 2009-09-01 CN CN2009100921462A patent/CN101633742B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101241124A (en) * | 2008-02-18 | 2008-08-13 | 博奥生物有限公司 | A kind of biochip substrate and preparation method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101633742A (en) | 2010-01-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP6279654B2 (en) | Polymer compound substrate having glass-like surface, and chip made of said polymer compound substrate | |
| Witters et al. | Biofunctionalization of electrowetting-on-dielectric digital microfluidic chips for miniaturized cell-based applications | |
| CN108165119A (en) | Polymer peridium | |
| CN109689216A (en) | The protection surface covering of flow cell | |
| Feyssa et al. | Patterned immobilization of antibodies within roll-to-roll hot embossed polymeric microfluidic channels | |
| CN103335984A (en) | Microarray chip without solid wall based on LSPR (Localized Surface Plasmon Resonance) and application thereof | |
| CN101371139A (en) | Supports for assaying analytes and methods of making and using thereof | |
| CN114555245B (en) | Amphiphobic surfaces with layered structures, methods of making and uses thereof | |
| Seguin et al. | Surface modification of poly (dimethylsiloxane) for microfluidic assay applications | |
| CN101315330B (en) | Preparation method of surface plasmon resonance imaging gold film dot microarray | |
| CN102242053A (en) | Biochip with polymer three-dimensional nanostructure | |
| CN108226259B (en) | Application of an ultra-wetting and highly sensitive electrochemical microchip as an electrochemical biosensor | |
| CN103233274A (en) | Preparation method of polymer based three-dimensional (3D) biochip | |
| CN106345544A (en) | Active amino-modified substrate for microarray and preparation method of active amino-modified substrate | |
| CN101633742B (en) | Aminoplast substrate and its preparation method and application | |
| CN102168146A (en) | High-specificity and high-sensitivity gene chip as well as preparation method and application thereof | |
| CN108855259A (en) | A kind of surface modifying method of micro-array chip | |
| CN102276863B (en) | Amino plastic substrate and preparation method and application thereof | |
| CA2245013C (en) | Analytical measurement method and its use | |
| CN101643321B (en) | High-polymer three-dimensional amino-group substrate as well as preparation method and application thereof | |
| CN108503565A (en) | A novel biochip substrate, its preparation method and application | |
| Raoof et al. | Improving the selectivity by using different blocking agents in DNA hybridization assays for SiGe bio-molecular sensors | |
| KR100766752B1 (en) | PNA chip using a polymer substrate coated with a polymer having an epoxy group | |
| KR20030061523A (en) | Oligomer for fixing biomolecule, and composition for fixing bio material comprising the same | |
| CN1818649A (en) | Agarose gel plastic substrate, its production and use |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C56 | Change in the name or address of the patentee |
Owner name: CAPITALBIO CORPORATION CO., LTD. Free format text: FORMER NAME: CAPITALBIO CORPORATION |
|
| CP01 | Change in the name or title of a patent holder |
Address after: 102206 Beijing City, Changping District Life Science Park Road No. 18 Patentee after: CAPITALBIO CORPORATION Patentee after: Tsinghua University Address before: 102206 Beijing City, Changping District Life Science Park Road No. 18 Patentee before: Capitalbio Corporation Patentee before: Tsinghua University |